Dye-labeled and polymerized antibody and method for preparing the same

ABSTRACT

The present invention provides a highly sensitive dye-labeled and polymerized antibody that can detect a target substance even when the target substance has a low concentration. The dye-labeled and polymerized antibody of the present invention comprises a polymerized antibody, which has been polymerized with a polyfunctional reagent, and a cyanine dye for labeling the polymerized antibody represented by the formula (1) given below:                    
     where R 1  and R 2  denote hydrogen or an alkyl group, X denotes a halogen, M denotes hydrogen or an alkali metal, and n represents an integer in a range of 1 to 4.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/831,204 filed Apr. 2, 1997, now U.S. Pat. No. 5,922,618, and acontinuation-in-part of application Ser. No. 08/745,337 filed Nov. 8,1996, now U.S. Pat. No. 5,965,713.

BACKGROUND OF THE INVENTION

The present invention relates to a polymerized antibody labeled with acyanine dye and a method for preparing the same.

The dye-labeled antibody prepared by labeling an antibody with a dyespecifically reacts with an antigen included in a sample solution and isreadily recognizable with naked eyes. The dye-labeled antibodies areaccordingly applied for immunosensors, each of which takes advantage ofan immunological antigen-antibody reaction to detect a target substanceincluded in a sample solution, and are used for diagnoses in a varietyof medical institutions.

Cyanine dyes having the high molar absorption coefficient and the highreactivity are often used to label antibodies (Bioconjugate ChemistryVol. 4, No. 2, pp105-111, 1993).

The functional group of the cyanine dye reacts with and is covalentlybound to an amino group or a carboxyl group included in an antibody, and20 to 50 molecules of the dye are attached to one molecule of theantibody.

The cyanine dye-labeled antibody thus prepared generally has high visualrecognizability, and is effectively applied for, for example,immunochromatography to detect a small amount of a specific substance,such as human chorionic gonadotropin (HCG) that is present only in theurine of pregnant women.

One molecule of the antibody generally has only two sites reacting withthe antigen and thereby has relatively low reaction sensitivity to theantigen.

When the conventional dye-labeled antigen is used for an immunosensor,the immunosensor accordingly does not have sufficient sensitivity. Inthe case that the sample solution contains a low concentration of atarget substance (antigen), it is difficult to detect the targetsubstance.

BRIEF SUMMARY OF THE INVENTION

The object of the present invention is thus to provide a highlysensitive dye-labeled and polymerized antibody that enables detection ofeven a low concentration of a target substance.

Another object of the present invention is to provide a method forpreparing such a dye-labeled and polymerized antibody.

The present invention provides a dye-labeled and polymerized antibodycomprising an antibody and a cyanine dye represented by the formula (1)given below:

where R₁ and R₂ denote hydrogen or an alkyl group, X denotes a halogen,M denotes hydrogen or an alkali metal, and n represents an integer in arange of 1 to 4,

wherein the antibody is polymerized via a polyfunctional reagent and thepolymerized antibody is labeled with the cyanine dye.

The polymerized antibody is a polyvalent antibody having a large numberof sites reacting with an antigen and thereby has a higher bindingsensitivity to the antigen, compared with the conventional divalentantibody.

The number of dye molecules bound to one molecule of the polymerizedantibody is greater than the number of dye molecules bound to onemolecule of the conventional antibody. This improves the visualrecognizability.

When the dye-labeled and polymerized antibody of the present inventionis applied for, for example, immunochromatography, theimmunochromatography can detect a target substance (sample) with highsensitivity even when the sample has a low concentration. Because of thehigh sensitivity, the dye-labeled and polymerized antibody of thepresent invention is applicable for biosensors.

In accordance with one preferable application of the dye-labeled andpolymerized antibody of the present invention, a skeleton of the cyaninedye is bound to the polymerized antibody via a covalent bond between anacyl carbon originated from a succinimidyl group in the cyanine dye anda nitrogen originated from an amino group in the polymerized antibody.

The degree of polymerization of antibody in the dye-labeled andpolymerized antibody of the present invention is generally in a range of2 to 50.

The present invention is also directed to a method for preparing adye-labeled and polymerized antibody. The method includes the steps of:polymerizing an antibody using a polyfunctional reagent in a neutral ora weak alkaline phosphate buffer solution; and adding a cyanine dyerepresented by the formula (1) given above to the buffer solution, so asto label the polymerized antibody. It is here preferable that thephosphate buffer solution has a pH value in a range of 7.0 to 8.0.

The antibody used to prepare the dye-labeled and polymerized antibody ofthe present invention is not specifically restricted, but may have avariety of origins and sub-classes. Available examples of the antibodyinclude immunoglobulins (Ig), such as mouse IgG, mouse IgM, mouse IgA,mouse IgE, rat IgG, rat IgM, rat IgA, rat IgE, rabbit IgG, rabbit IgM,rabbit IgA, rabbit IgE, goat IgG, goat IgM, goat IgE, goat IgA. sheepIgG, sheep IgM, sheep IgA, and sheep IgE. These antibodies may be ofcommercial origin or directly collected from the corresponding animals.

The polyfunctional reagent used in the present invention has two or morefunctional groups (for example, succinimidyl group, pyridyldisulfidegroup), which can be linked with an antibody, in one identical molecule.Available examples of the polyfunctional reagent includedithiobis(sulfosuccinimidyl propionate) represented by the formula (2),bis (sulfosuccinimidyl) suberate represented by the formula (3),disuccinimidyl tartrate represented by the formula (4), ethylene glycolbis(succinimidyl succinate) represented by the formula (5), andN-succinimidyl-3-(2-pyridyldithio) propionate represented by the formula(6).

The cyanine dye represented by the formula (1) is a red dye readilyrecognizable with naked eyes. The cyanine dye has a less number ofconjugated carbons and thereby has the highest solubility in water amonga variety of cyanine dyes.

The halogen represented by X in the formula (1) may be fluorine,chlorine, bromine, or iodine. The metal represented by M may be lithium,sodium, or potassium.

While the novel features of the invention are set forth particularly inthe appended claims, the invention, both as to organization and content,will be better understood and appreciated, along with other objects andfeatures thereof, from the following detailed description taken inconjunction with the drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is a perspective view schematically illustrating the structure ofan immunochromatography sensor as an example of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following describes one exemplified process of synthesizing thecyanine dye represented by the formula (1) given above.

The process first dissolves hydrazinobenzenesulfonic acid (7) andisopropyl methyl ketone in an acidic solvent and heats the mixture toobtain indoleniumsulfonate (8). The process then adds a metalhydroxide-saturated alcohol solution into an alcohol solution ofindoleniumsulfonate (8), so as to yield a metal salt ofindoleniumsulfonate (9).

The process subsequently adds a halogenized fatty acid to an organicsolvent solution of the metal salt (9) and heats the mixture to obtain ametal salt of carboxyalkylindoleniumsulfonate (10). By taking intoaccount the solubility in water, it is preferable that the halogenizedfatty acid has one to four carbon atoms.

The process then dissolves the metal salt (10) and N-carboxyethyl-3,3-dimethylindolenine into a basic organic solvent and heats the mixtureto prepare a carboxylic acid derivative (11). The process subsequentlyadds hydroxysuccinimide and dicyclohexylcarbodiimide as a condensingagent to the organic solvent solution of the carboxylic acid derivative(11) and well stirs the mixture to yield the cyanine dye represented bythe formula (1).

The halogen included in the respective compounds represented by theformula (1), the formula (10), and the formula (11) may be fluorine,chlorine, bromine, or iodine. The metal included in the respectivecompounds represented by the formula (1) and the formulae (9) through(11) may be lithium, sodium, or potassium.

The following describes the mechanism of polymerization reaction of theantibody with the polyfunctional reagent.

When the antibody is mixed with the polyfunctional reagent(dithiobis(sulfosuccinimidyl propionate) having two or more succinimidylgroups) as shown by the formula (12), an amino group in the antibodyapproaches an ester bond of one succinimidyl group in the reagent asshown by the formula (13).

The amino group reacts with the ester bond as shown by the formula (14),so that one hydrogen atom is released from the amino group. The hydrogenatom released from the amino group is attached to succinimide in thesuccinimidyl group. Succinimide is then changed to hydroxysuccinimide,which is released from the succinimidyl group. At the same time, theresidue of the succinimidyl group and the hydrogen atom-released aminogroup combine to form an amide bond, through which the reagent is linkedwith the antibody.

The other succinimidyl groups in the reagent are subjected to thesimilar reaction, so that the reagent is bound to other antibodiesthrough the amide bond as shown by the formula (15). This reaction isrepeated to polymerize the antibody.

The succinimidyl group in the cyanine dye is bound to the amino group inthe antibody according to the same mechanism as discussed above.

The present invention is described more in detail with a concreteexample.

(1) Polymerization of Mouse IgG

The polymerization process first dissolved 10 mg (6.667×10⁻⁵ mmol) ofmouse IgG (hereinafter simply referred to as IgG) into 1 ml of aphosphate buffer solution (hereinafter referred to as PBS). The processthen added dropwise 0.1 ml of a PBS solution ofdithiobis(sulfosuccinimidyl propionate) (manufactured by Pierce Corp.,hereinafter referred to as DTSSP) with stirring at room temperature. ThePBS solution of DTSSP added dropwise contained 4.057 mg (0.006667 mmol,100 equivalents) of DTSSP.

After stirring the mixed solution at 35° C. for 30 minutes, the processfiltered the mixed solution through a Sepharose gel (manufactured byPharmacia Fine Chemical Inc., Sephadex G25M column). This gaveapproximately 6 ml of the PBS solution containing IgG aggregate(hereinafter referred to as IgGagg.). The concentration of the PBSsolution thus obtained was determined by the procedure discussed below.

The procedure collected 0.5 ml of the PBS solution and measured theabsorbance at 280 nm. The observed absorbance was 2.43. The absorbanceat 280 nm is attributed to IgG, so that the concentration [IgGagg.] ofIgG molecules in the IgG aggregate is determined according to thefollowing equation. Here the molar absorption coefficient of IgG at 280nm is set equal to 2.099×10⁵.

[IgGagg.] =2.43/2.099×10⁵=1.158×10⁻⁵(M)

(2) Labeling Polymerized Antibody with Dye

The process dissolved the cyanine dye represented by the formula (1)into 0.2 ml of the PBS (400 equivalents of the total protein quantity)to obtain 26.8 mg of a dye solution (hereinafter referred to as SLIC1).The cyanine dye included iodine as X, potassium as M, and 2 carbon atomsas n in the formula (1)

The process slowly added the SLIC1 dropwise to the IgGagg. solution(total amount of antibody: 10 mg) obtained in the process (1). After themixed solution was stood still at 4° C. for 20 hours, the processdialyzed the mixed solution against 20 liters of a PBS solutioncontaining sodium azide as an antiseptics, in order to remove unreacteddye molecules. This gave approximately 6 ml of the PBS solutioncontaining SLIC1-labeled polymerized antibody.

The number of SLIC1 molecules per one IgG molecule in the SLIC1-labeledpolymerized antibody was determined according to the followingprocedure.

The observed absorbance of the resultant solution was 6.80 at 280 nm and40.2 at 430 nm. The polymerized antibody does not have absorption at 430nm, so that the observed absorbance at 430 nm is attributed to SLIC1.The concentration [SLIC1] of the SLIC1 is thus determined by thefollowing equation. Here the molar absorption coefficient of SLIC1 at430 nm is set equal to 1×10⁵.

[SLIC1]=40.2/1×10⁵ ⁼4.02×10⁻⁴(M)

The observed absorbance at 280 nm is originated from IgG of thepolymerized antibody. The bound SLIC1, however, also has absorption at280 nm. The concentration [IgGagg.] of the IgG molecules in thepolymerized antibody is accordingly determined by subtracting the effectof this absorption. Here Ab_(280.IgG) represents the absorbanceattributed to the polymerized antibody at 280 nm, the molar absorptioncoefficient of SLIC1 at 280 nm is set equal to 9.8×10³, and the molarabsorption coefficient of the IgG molecules in the polymerized antibodyat 280 nm is 2.099×10⁵.

Ab_(280.IgG)=6.8−(4.02×10⁻⁴×9.8×10³)=2.86

[IgGagg.]=2.86/2.099×10⁵=1.363×10⁻⁵(M)

The number of SLIC1 molecules bound to one IgG molecule in theSLIC1-labeled polymerized antibody is accordingly given by:

[SLIC1]/[IgGagg.]=4.02×10⁻⁴/1.363×10⁻⁵=29.5

(3) Evaluation of Dye-Labeled and Polymerized Antibody

The dye-labeled and polymerized antibody obtained in the process (2) wasapplied for an immunochromatography sensor. The luminescence due toaggregation of the dye-labeled and polymerized antibody was determinedby measuring the absorbance at 430 nm.

FIG. 1 is a perspective view schematically illustrating the structure ofthe immunochromatography sensor. A first glass filter 2, anitrocellulose antibody fixation film 5, and a second glass filter 6 arearranged in this sequence on a plate base 1 made of plastics, such aspoly(vinyl chloride). One end of the first glass filter 2 that is incontact with the antibody fixation film 5 is impregnated with thedye-labeled and polymerized antibody obtained in the process (2). Thisend forms a labeled antibody section 3. An antibody that reacts with thesame antigen as the dye-labeled and polymerized antibody is fixed byadsorption on a predetermined area of the antibody fixation film 5. Thisarea forms an antibody fixation section 4.

The absorbance is measured by the following procedure with theimmunochromatography sensor having the above structure.

Referring to FIG. 1, when a sample solution is added dropwise to theother end of the first glass filter 2 that is not in contact with theantibody fixation film 5, the sample solution moves from the first glassfilter 2 towards the second glass filter 6 based on the principle ofchromatography. On the labeled antibody section 3, the dye-labeled andpolymerized antibody is bound to the antigen included in the samplesolution. The sample solution containing the antigen bound to thelabeled antibody then moves to the antibody fixation section 4, wherethe antigen is bound to the fixation antibody and fixed. The remainingsample solution continues moving across the antibody fixation film 5 andis eventually absorbed by the second glass filter 6.

The absorbance was determined by irradiating the antibody fixationsection 4 with light (L1) having the wavelength of 430 nm and measuringreflected light L2.

A dye-labeled antibody of comparative example was prepared in the samemanner as above, except the polymerization of the antibody. Thedye-labeled antibody of comparative example was also applied forimmunochromatography sensor, and the absorbance was determined by theabove procedure.

The absorbance of the dye-labeled and polymerized antibody of thepresent invention was about 0.8, whereas the absorbance of thedye-labeled antibody of comparative example was about 0.07.

This result clearly shows that the sensitivity of the dye-labeled andpolymerized antibody of the present invention is approximately 10 timesthe sensitivity of the comparative example.

As described above, the dye-labeled and polymerized antibody of thepresent invention has a large number of reaction sites with an antigenand thereby has high sensitivity to the antigen. The dye-labeled andpolymerized antibody of the present invention is, for example,effectively applied for immunochromatographysensor. This sensor has thesignificantly higher sensitivity than that of a sensor with a labeledantibody prepared by the conventional method.

Although the present invention has been described in terms of thepresently preferred embodiments, It is to be understood that suchdisclosure is not to be interpreted as limiting. Various alterations andmodifications will no doubt become apparent to those skilled in the artto which the present invention pertains, after having read the abovedisclosure. Accordingly, it is intended that the appended claims beinterpreted as covering all alterations and modifications as fall withinthe true spirit and scope of the invention.

What is claimed is:
 1. A dye-labeled and polymerized antibody comprisingan antibody and a cyanine dye represented by the following formula (1):

where R₁ and R₂ denote hydrogen or an alkyl group, X denotes a halogen,M denotes hydrogen or an alkali metal, and n represents an integer in arange of 1 to 4, wherein said antibody is polymerized via apolyfunctional reagent and the polymerized antibody is labeled with saidcyanine dye.
 2. The dye-labeled and polymerized antibody in accordancewith claim 1, wherein a skeleton of said cyanine dye is bound to saidpolymerized antibody via a covalent bond between an acyl carbonoriginated from a succinimidyl group in said cyanine dye and a nitrogenoriginated from an amino group in said polymerized antibody.
 3. A methodfor preparing a dye-labeled and polymerized antibody, said methodcomprising the steps of: polymerizing an antibody using a polyfunctionalreagent in a neutral or a weak alkaline phosphate buffer solution; andadding a cyanine dye represented by the following formula (1) to saidbuffer solution to label the polymerized antibody:

where R₁ and R₂ denote hydrogen or an alkyl group, X denotes a halogen,M denotes hydrogen or an alkali metal, and n represents an integer in arange of 1 to 4.